Acid-stable alpha-amylases from fungal origin have been discovered a long time ago. For example, the acid-stable alpha-amylase from A. niger has been known for over 30 years (Y. Minoda, K. Yamada, Agr. Biol. Chem., 27(11), 806-811(1963)) and has been thoroughly characterised by Minoda, Yamada and co-workers. The same authors showed the stabilisation of the enzyme activity in the presence of Ca.sup.2+. The presence of an acid-unstable alpha-amylase in the Aspergillus niger preparations was disclosed by the same authors. The acid-stable alpha-amylase enzyme has a pH optimum between 3 and 4 and the optimal temperature is in the range of 70 to 75.degree. C.
Several procedures have been developed for obtaining acid-stable alpha-amylase in purified form. One method (Minoda and Yamada, cited above) uses fractional precipitation with ammonium sulfate, rivanol and acetone to obtain the enzyme in crystalline form. It was found that this crystalline alpha-amylase was contaminated with an acid-unstable alpha-amylase, whereas glucoamylase and transglucosidase were found to be removed. The acid-unstable alpha-amylase could subsequently be removed by treatment of the alpha-amylase mixture at acidic pH (pH 2.5) and 37.degree. C., followed by fractionation. A further purification was performed by recrystallisation with acetone and gelfiltration with Sephadex G-50.
Other publications describe a purification method based on chromatography on DEAE-Sephadex A-25 (D. S. Chong, Y. Tsujisaka, J. Fennent. Technol, 54(4), 264-266(1976)) or ammonium sulfate precipitation followed by chromatography on DEAE-Sephadex A-50 (N. Ramasesh, K. R. Sreekantiah, V. S. Murthy, Starch, 34(8), 274-279(1982)). Also Sephadex G-25, followed by Sepharose Q Fast Flow chromatography has been utilised (Y-Y. Linko, X. Y. Wu, Biotechnology Techniques, 7(8)).
European patent application EP 0138 428 describes the production of an alpha-amylase which is free from transferase and amyloglucosidase. This is achieved by treating suitable Aspergillus niger with mutagenic agents and selecting mutant strains which do not produce the undesired enzyme activities. The fermentation broth of these mutant strains then mainly contains the acid-stable alpha-amylase activity.
The dextrinisation and saccharification properties of the acid-stable alpha-amylase from A. niger have been extensively reported. The conversion of liquefied starch into glucose using a blend of glucoamylase and acid-stable alpha-amylase is described in European patent application 0,140,410 where it is shown that the presence of acid-stable alpha-amylase shortens the saccharification time and gives higher dextrose yields. Also the paper of Linko et al.(cit. above) describes the use of acid-stable alpha-amylase in the production of dextrose.
Hansen (T. T. Hansen, New Approaches to Research on Cereal Carbohydrates, eds. R. D Hill and L. Munck, Elsevier Science Publishers B. V., Amsterdam 1985, 211-216) has reported the use of acid-stable alpha-amylase for the saccharification of a 12 DE maltodextrin, to obtain a syrup with 7% glucose, 48% DP2, 26% DP3 and 20% DP4+ after 96 hours. The production of a 63 DE syrup out of a 42 DE acid liquefied syrup by an immobilized acid-stable alpha-amylase has been described in the same paper. This paper however does not describe how the acid-stable alpha-amylase which is used was purified. DE (dextrose equivalent) is a measure for the number of reducing groups which are present in the molecules. Pure glucose has a DE of 100 and undegraded starch has a DE of 0. Liquefaction of starch with the acid-stable alpha-amylase at 75-85.degree. C. gave starch substrates, which were saccharified by glucoamylase. The obtained syrups had the correct dextrose yield, but were starch positive and had poor filterabilities. The use of immobilised acid-stable alpha-amylase obtained from mutant strains has been described in European patent application EP 0,157,638.
With the potential to be used as a post-liquefaction enzyme for high maltose production where a low initial DE is required, a starch slurry pre-liquefied with B. Iicheniformis alpha-amylase was subjected to incubation with acid-stable alpha-amylase at 90.degree. C. and pH 5. After 20 minutes the temperature was lowered to 60.degree. C. and barley beta-amylase together with pullulanase was added. The final syrup contained 0.5% glucose and 71% maltose and 91% fermentable sugars. A reference syrup produced without alpha-amylase had the same DP1 and DP2 composition, but only 77% fermentable sugars.
An acid-stable alpha-amylase from A. niger able to degrade raw starch has been described (B. N. Okolo, L. I. Ezeogu, C. N. Mba, J. Sci. Food. Agric., 69,109-115 (1995)). Acid-stable alpha-amylases can not only be found in Aspergillus niger, also in Aspergillus awamori acid stable alpha-amylases have been found (R. S. Bhella, I. Altosaar, Can. J. Microbiol., 31, 149-153(1985)) these alpha-amylases were reported to be stable between pH 3.5 and 6.5.
Although it is apparent from the mentioned literature that the acid-stable alpha-amylase from Aspergillus niger, or from other Aspergillus species, which produce acid-stable alpha-amylase, is of potential industrial significance, no commercial Aspergillus sp. acid-stable alpha-amylase preparation is free of glucoamylase side activity. The presence of the glucoamylase in the cornmercial acid-stable alpha-amylase preparations significantly reduces the range of applications where these preparations can be utilized.